Fluctuating asymmetry (FA) is the small random deviation from perfect symmetry in bilateral traits and is often used to assess developmental instability (DI) experienced by organisms. In this study, with a unique dataset of 1389 deceased human fetuses, we investigated the relationship between abnormal development and human limb FA in different ways, using a more fundamental approach than usually done. We studied whether there is an underlying developmental basis of DI, as measured by FA, by investigating, first, whether limb FA can be attributed to developmental abnormalities associated with specific organ systems, germ layers or patterning processes, and second, whether limb FA increases with increasing number of developmental abnormalities either gradually, or in a threshold-like fashion. Limb FA was found to increase in fetuses with cardiovascular and nervous system abnormalities. Fetuses with ectoderm-derived abnormalities were also found to have significantly higher limb FA, but no other germ layers were found to be associated. We found no significant correlation between specific developmental processes, such as neural crest development, segmentation, midline and left-right patterning and limb FA. Although only some congenital abnormalities were correlated with limb FA, our results do suggest that limb FA increases when an increasing number of organ systems, germ layers or developmental pathways are disrupted. Therefore, we conclude that limb FA is mainly a good indicator for DI in the case of particularly severe perturbations of development and that FA does not reflect the subtler deviations from developmental stability.